Filoviruses have been associated with episodic but increasingly frequent outbreaks of a highly lethal hemorrhagic fever, and are agents of concern for bioterrorism. No FDA-approved vaccines or drugs are currently available to prevent or treat filovirus infections. Antiviral drugs targeting host components essential for viral infection have the potential to confer broad protection against viruses with similar infection pathways, and they may be less likely to engender drug resistance than their counterparts targeting viral components. Unfortunately, few host factors critical for filovirus infection have been identified. However, we recently reported that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter expressed in all cells, is broadly required by filoviruses for entry and infection of human cells, and for pathogenesis in mice. We also found that a single luminal segment (domain C) in NPC1 mediates filovirus entry by binding specifically and directly to filovirus GP. Therefore, the GP-NPC1 interaction represents a new and unique molecular target for developing broadly active anti-filovirus therapeutics. Our overall goals are to identify small molecules targeting the critical interaction between the envelope glycoprotein (GP) and NPC1, and to develop them into clinical candidates with broad-spectrum activity against filoviruses. The major milestone of this proposal is to select primary and backup pre-clinical candidates suitable for anti-filovirus IND-enabling studies. To fulfill these objectives, the PI has assembled a multidisciplinary team that includes a company with demonstrated success in the discovery and development of anti-infectives and a virologist who brings longstanding experience in the investigation of filoviruses under BSL-4 containment. Together, we will: 1. Develop assays to screen for inhibitors targeting the GP-NPC1 interaction 2. Perform high-throughput chemical screens to identify and confirm GP-NPC1 interaction inhibitors 3. Validate the antiviral activity o hits and early leads and elucidate their mechanisms of action 4. Chemically optimize validated hits for antiviral activity and in vitro pharmacology 5. Establish pharmacologic and toxicological profiles of lead inhibitors and evaluate them for antiviral efficacy in murine infection models